Oligonucleotides for controlling amplification of nucleic acids

ABSTRACT

Methods and oligonucleotides are provided for detecting an internal control nucleic acid for qualitative and/or quantitative purposes.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 14/837,931, filed on Aug. 27, 2015, which claims the benefit under35 U.S.C. § 119 of European Patent Application No. 14182730.3 filed onAug. 28, 2014. The entire disclosures of the above-referenced priorapplications are hereby incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named “32866_Sequence_Listing.txt”, having a size in bytes of4 kb, and created on Aug. 28, 2014. The information contained in thiselectronic file is hereby incorporated by reference in its entiretypursuant to 37 CFR § 1.52(e)(5).

FIELD OF THE INVENTION

The present invention belongs to the field of in-vitro diagnostics.Within this field, it particularly concerns the amplification anddetection of a control nucleic acid for qualitative and/or quantitativepurposes.

BACKGROUND

In the field of molecular diagnostics, the amplification of nucleicacids from numerous sources has been of considerable significance.Examples for diagnostic applications of nucleic acid amplification anddetection are the detection of viruses such as Human Papilloma Virus(HPV), West Nile Virus (WNV) or the routine screening of blood donationsfor the presence of Human Immunodeficiency Virus (HIV), Hepatitis-B(HBV) and/or C Virus (HCV). Furthermore, said amplification techniquesare suitable for bacterial targets such as mycobacteria, or the analysisof oncology markers.

The most prominent and widely-used amplification technique is PolymeraseChain Reaction (PCR). Other amplification reactions comprise, amongothers, the Ligase Chain Reaction, Polymerase Ligase Chain Reaction,Gap-LCR, Repair Chain Reaction, 3 SR, NASBA, Strand DisplacementAmplification (SDA), Transcription Mediated Amplification (TMA), andQ-amplification.

Automated systems for PCR-based analysis often make use of real-timedetection of product amplification during the PCR process in the samereaction vessel. Key to such methods is the use of modifiedoligonucleotides carrying reporter groups or labels.

It has been shown that amplification and detection of more than onetarget nucleic acid in the same vessel is possible. This method iscommonly termed “multiplex” amplification and requires different labelsfor distinction if real-time detection is performed.

It is mostly desirable or even mandatory in the field of clinicalnucleic acid diagnostics to control the respective amplification usingcontrol nucleic acids with a known sequence, for qualitative(performance control) and/or quantitative (determination of the quantityof a target nucleic using the control as a reference) purposes. Giventhe diversity especially of diagnostic targets, comprising prokaryotic,eukaryotic as well as viral nucleic acids, and given the diversitybetween different types of nucleic acids such as RNA and DNA, controlnucleic acids are usually designed in a specific manner.

EP 2759604 discloses a method for detection of a set of non-competitiveinternal control nucleic acids.

Described herein are improved oligonucleotides and methods for thispurpose.

DESCRIPTION

A first aspect described herein is an isolated oligonucleotide with thesequence of SEQ ID NO 1. This oligonucleotide can be used as a probe forthe improved detection of a control nucleic acid comprising the sequenceof SEQ ID NO 4.

Thus, another aspect described herein is a use of a probe with SEQ ID NO1 for the detection of a control nucleic acid comprising SEQ ID NO 4.

The sequence of SEQ ID NO 4 is a scrambled sequence not showing anysignificant homology to any naturally occurring sequences, such that itmay serve as a non-competitive internal control nucleic acid sequencefor qualitative and/or quantitative detection of multiple target nucleicacids, as described in EP 2759604.

In nucleic acid amplification assays such as, for instance, PCR assayslike real-time PCR assays, SEQ ID NO 4 can be efficiently amplified witha specific set of primers comprising a first primer with SEQ ID NO 2 anda second primer with SEQ ID NO 3.

Hence, an aspect described herein is a composition comprising theoligonucleotides with the SEQ ID NOs 1, 2 and 3.

These nucleic acids may be provided to the skilled person as akit-of-parts, further comprising a suitable amplification and detectiontarget, namely a control nucleic acid with the sequence of SEQ ID NO 4.

Therefore, a further aspect described herein is a kit for controllingthe detection of multiple target nucleic acids, the kit comprising adetection probe with SEQ ID NO 1, a pair of amplification primers withSEQ ID NO 2 and SEQ ID NO 3, and a control nucleic acid comprising SEQID NO4.

The use of a control nucleic acid comprising the sequence of SEQ ID NO 4with the improved detection by a probe with SEQ ID NO 1 as describedherein facilitates the development of improved simultaneous assays on aplurality of parameters and/or nucleic acid types while using the sameinternal control nucleic acid sequence for said different parametersand/or nucleic acid types. Therefore, it contributes to reducing theoverall complexity of the corresponding experiments on various levels:For instance, only one internal control nucleic acid sequence has to bedesigned and added to the respective amplification mixes, thus savingthe time and costs for designing and synthesizing or buying multiplecontrol nucleic acid sequences. The assay or assays can be streamlined,and the risk of handling errors is reduced. In addition, the moredifferent control nucleic acid sequences are employed in one assay orparallel assays that may be carried out simultaneously under the sameconditions, the more complex it may result to adjust the respectiveconditions. Moreover, with a single control suitable for a plurality ofnucleic acids, said control can be dispensed from a single source intodifferent vessels containing said different target nucleic acids. Insome embodiments, the single control nucleic acid sequence may alsoserve as a qualitative and as a quantitative control.

The improvement of the detection of a control nucleic acid comprisingSEQ ID NO 4 by using a probe with the sequence of SEQ ID NO 1, as shownin the Example herein, enhances the reliability of the measurementscarried out according to the method described in EP 2759604. The probewith the sequence of SEQ ID NO 1 does not overlap with anyoligonucleotide sequences disclosed in EP 2759604, in particular notwith the probe with the sequence of SEQ ID NO 5 (disclosed as SEQ ID NO52 in EP 2759604) against which SEQ ID NO 1 has been tested in thecurrent Example.

A process in which the use of a control nucleic acid comprising thesequence of SEQ ID NO 4 with the improved detection by a probe with SEQID NO 1 can be advantageously applied is the following:

A process for internally controlled isolating and simultaneouslyamplifying a first and a second target nucleic acid that may be presentin one or more fluid samples, said process comprising the automatedsteps of:

-   -   a. adding an internal control nucleic acid comprising the        sequence of SEQ ID NO 4 to each of said fluid samples    -   b. combining together a solid support material and said one or        more fluid samples in one or more vessels for a period of time        and under conditions sufficient to permit nucleic acids        comprising the target nucleic acids, if present on said one or        more fluid samples, and the internal control nucleic acid to be        immobilized on the solid support material    -   c. isolating the solid support material from the other material        present in the fluid samples in a separation station    -   d. purifying the nucleic acids in said separation station and        washing the solid support material one or more times with a wash        buffer    -   e. contacting the purified target nucleic acids and the purified        internal control nucleic acid with amplification reagents        comprising a distinct set of primers and a probe for each of        said target nucleic acids and a set of primers comprising a        first primer with SEQ ID NO 2 and a second primer with SEQ ID NO        3 and a probe with SEQ ID NO 1 for said internal control nucleic        acid in at least two reaction vessels, wherein a first reaction        vessel comprises primers and a probe for said first target        nucleic acid and at least a second reaction vessel comprises        primers and a probe for said second target nucleic acid and        wherein the primers and the probe for the first target nucleic        acid are absent from the second reaction vessel and the primers        and the probe for the second target nucleic acid are absent from        the first reaction vessel    -   f. incubating in said reaction vessels said purified target        nucleic acids and said purified internal control nucleic acid        with said amplification reagents for a period of time and under        conditions sufficient for an amplification reaction indicative        of the presence or absence of said target nucleic acids to occur    -   g. detecting and measuring signals generated by the        amplification products of said target nucleic acids and being        proportional to the concentration of said target nucleic acids,        and detecting and measuring a signal generated by said internal        control nucleic acid,    -   wherein the conditions for amplification and detection in        steps d. to g. are identical for the first and the second        reaction vessel and thus for the target nucleic acids and the        internal control nucleic acid.

As one of the advantages of the process described herein, the testing ofa particular biological sample for other nucleic acids in possiblesubsequent experiments need not involve another sample preparationprocedure with the addition of a different internal control nucleicacid, since the control comprising SEQ ID NO 4 can be used to controlthe amplification of different nucleic acids. Thus, once an internalcontrol nucleic acid has been added, other parameters may be tested inthe same sample under the same conditions.

The internal control nucleic acid described above is non-competitive.

A “non-competitive internal control nucleic” acid has different primerbinding sites than the target and thus binds to different primers.Advantages of such a setup comprise, among others, the fact that thesingle amplification events of the different nucleic acids in thereaction mixture can take place independently from each other withoutany competition effects. Thus, no adverse effects occur regarding thelimit of detection of the assay as can be the case in a competitivesetup.

The fact that the process described herein involves a distinct set ofprimers for each of said target nucleic acids and for said internalcontrol nucleic acid renders the method considerably flexible. In thisnon-competitive setup it is not necessary to introduce target-specificbinding sites into the control nucleic acid as in the case of acompetitive setup, and the drawbacks of a competitive setup, such ascompetition for amplification reagents, are avoided. In anon-competitive setup, the internal control nucleic acid has a sequencedifferent from any target sequences, in order not to compete for theirprimers and/or probes. The sequence of the internal control nucleic acidis different from the other nucleic acid sequences in the fluid sample.As an example, if the fluid sample is derived from a human, the internalcontrol nucleic acid does not have a sequence which also endogenouslyoccurs within humans. The difference in sequence is thus at leastsignificant enough to not allow the binding of primers and/or probes tothe respective endogenous nucleic acid or acids under stringentconditions and thus render the setup competitive. SEQ ID NO 4 is ascrambled sequence originally based on a naturally occurring genome. Asknown in the art, “scrambling” means introducing a number of basemutations into a sequence. In the case of SEQ ID NO 4, the sequence ofthe internal control nucleic acid used in the invention is substantiallyaltered with respect to the naturally occurring gene it is derived from.

The process comprising the automated steps as described herein alsodisplays various additional advantages:

It has been a challenge in the prior art that the number of differenttarget nucleic acids in a multiplex assay carried out in a singlereaction vessel is limited by the number of appropriate labels. In areal-time PCR assay, for example, the potential overlap of fluorochromespectra has a great impact on assay performance (risk of false positiveresults, lower precision etc.) Therefore, the respective fluorophoreshave to be carefully selected and spectrally well separated in order toassure the desired performance of a diagnostic test. Typically, thenumber of different usable fluorophores corresponds to a single-digitnumber of PCR instrument fluorescence channels.

In contrast, in the process described herein, the internally controlledamplification of at least a first and a second target nucleic acid takesplace in at least two different reaction vessels, allowing for thesimultaneous amplification of a higher number of different targetnucleic acids, since signals in different reaction vessels can bedetected independently from each other. Still, included herein areembodiments wherein in one or more of the multiple reaction vesselsmultiplex reactions are performed, thereby multiplying the number oftargets that may be amplified simultaneously and under the sameconditions. In such embodiments, the internal control nucleic acidserves as a control for the different target nucleic acids within avessel as well as different target nucleic acids in different vessel.

Thus, in some embodiments of the process described herein, at least twotarget nucleic acids are amplified in the same reaction vessel.

Especially if a fluid sample is suspected to contain target nucleicacids from different organisms, or even the different organisms as such,or if it is not clear which of the different nucleic acids or organismsmay be present in said sample, an embodiment is the process describedherein, wherein the first target nucleic acid and the second targetnucleic acid are from different organisms.

In an embodiment of the process described herein, the first and/or thesecond target nucleic acid is a viral nucleic acid.

In a further embodiment of the process described herein, the firstand/or the second target nucleic acid is a non-viral nucleic acid.

In yet a further embodiment of the process described herein, the firstand/or the second target nucleic acid is a bacterial nucleic acid.

As described before, the process described herein is useful forqualitatively or quantitatively controlling the amplification of atleast a first and a second target nucleic acid.

Qualitative detection of a nucleic acid in a biological sample is, forinstance, crucial for recognizing an infection of an individual.Thereby, one important requirement for an assay for detection of amicrobial infection is that false-negative or false-positive results beavoided, since such results would almost inevitably lead to severeconsequences with regard to treatment of the respective patient. Thus,especially in PCR-based methods, a qualitative internal control nucleicacid is added to the detection mix. Said control is particularlyimportant for confirming the validity of a test result: At least in thecase of a negative result with regard to the respective target nucleicacid, the qualitative internal control reaction has to perform reactivewithin given settings, i.e. the qualitative internal control must bedetected, otherwise the test itself is considered to be inoperative.However, in a qualitative setup, said qualitative internal control doesnot necessarily have to be detected in case of a positive result. Forqualitative tests, it is especially important that the sensitivity ofthe reaction is guaranteed and therefore strictly controlled As aconsequence, the concentration of the qualitative internal control mustbe relatively low so that even in a situation of slight inhibition thequalitative internal control is not detected and therefore the test isinvalidated.

Thus, in some embodiments of the process described herein, the presenceof an amplification product of said internal control nucleic acid isindicative of an amplification occurring in the reaction mixture even inthe absence of amplification products for one or more of said targetnucleic acids.

On the other hand and in addition to mere detection of the presence orabsence of a target nucleic acid in a sample, it is often important todetermine the quantity of said nucleic acid. As an example, stage andseverity of a viral disease may be assessed on the basis of the viralload.

Further, monitoring of any therapy requires information on the quantityof a pathogen present in an individual in order to evaluate thetherapy's success. For a quantitative assay, it is necessary tointroduce a quantitative standard nucleic acid serving as a referencefor determining the absolute quantity of a target nucleic acid.Quantitation can be effectuated either by referencing to an externalcalibration or by implementing an internal quantitative standard.

In the case of an external calibration, standard curves are created inseparate reactions using known amounts of identical or comparablenucleic acids. The absolute quantity of a target nucleic acid issubsequently determined by comparison of the result obtained with theanalyzed sample with said standard function. External calibration,however, has the disadvantage that a possible extraction procedure, itsvaried efficacy, and the possible and often not predictable presence ofagents inhibiting the amplification and/or detection reaction are notreflected in the control.

This circumstance applies to any sample-related effects. Therefore, itmight be the case that a sample is judged as negative due to anunsuccessful extraction procedure or other sample-based factors, whereasthe target nucleic acid to be detected and quantified is actuallypresent in the sample.

For these and other reasons, an internal control nucleic acid added tothe test reaction itself is of advantage. When serving as a quantitativestandard, said internal control nucleic acid has at least the followingtwo functions in a quantitative test:

i) It monitors the validity of the reaction.

ii) It serves as reference in titer calculation thus compensating foreffects of inhibition and controlling the preparation and amplificationprocesses to allow a more accurate quantitation.

Therefore, in contrast to the qualitative internal control nucleic acidin a qualitative test which must be positive only in a target-negativereaction, the quantitative control nucleic acid in a quantitative testhas two functions: reaction control and reaction calibration. Thereforeit must be positive and valid both in target-negative andtarget-positive reactions.

It further has to be suited to provide a reliable reference value forthe calculation of high nucleic acid concentrations. Thus, theconcentration of an internal quantitative control nucleic acid needs tobe relatively high.

Therefore, in some embodiments, the process described herein furthercomprises the following step:

-   -   h. determining the quantity of one or more of said target        nucleic acids.

The internally controlled process described herein requires considerablyless hands-on time and testing is much simpler to perform than, forexample, real-time PCR methods used in the prior art. The process offersa major advantage in the field of clinical virology as it permitsparallel amplification of nucleic acids from several viruses like DNAand RNA viruses, bacteria, and/or other pathogens in parallelexperiments. The process is particularly useful in the management ofpost-transplant patients, in whom frequent viral monitoring is required.Thereby said process facilitates cost-effective diagnosis andcontributes to a decrease in the use of antiviral agents and in viralcomplications and hospitalizations. This equally applies to the field ofclinical microbiology. In general, efficiencies will be gained in fasterturnaround time and improved testing flexibility. Consequently, thisleads to a decrease in the number of tests requested on a patient tomake a diagnosis, and potentially shorter hospital stays (for example,if a diagnosis can be provided sooner, patients requiring antimicrobialtherapy will receive it sooner and thus recover earlier). In addition,patients show less morbidity and therefore cause fewer costs related tosupportive therapy (for example, intensive care related to a delay indiagnosis of sepsis).

Providing a negative result sooner can have important implications forthe overprescription of antibiotics. For example, if a test resultobtained by the process according to the invention is able to rule outthe pathogen more quickly than with a standard real-time PCR method,then the clinician will not be forced to use empirical antibiotics.Alternatively, if empirical antibiotics are used, the duration of therespective treatment can be shortened.

With respect to designing a specific test based on the process describedherein, the skilled artisan will benefit from the following advantages:

-   -   a reduction in software complexity (leading to a reduced risk of        programming errors)    -   focusing of assay development efforts on the chemistry        optimization instead of the chemistry plus the instrument        control parameters    -   much more reliable system since a single process is always used        and the hardware can be optimally designed to perform this        protocol    -   the skilled artisan performing the internally controlled process        described above is provided with the flexibility to run multiple        different assays in parallel as part of the same process    -   cost reduction.

In the context described herein, the term “solid support” as used hereinrelates to any type of solid support to which the analyte is capable ofbinding, either directly and non-specifically by adsorption, orindirectly and specifically. Indirect binding may be by binding to acapture nucleic acid probe which is homologous to a target sequence ofthe nucleic acid of interest. Thus, using capture probes attached on asolid support, a target nucleic acid can be separated from non-targetmaterial, or non-target nucleic acid. Such a capture probe isimmobilized on the solid support. Solid support material may be apolymer, or a composition of polymers. Other types of solid supportmaterial include magnetic silica particles, metal particles, magneticglass particles, glass fibers, glass fiber filters, filter paper etc.,while the solid support material is not limited to these materials.

“Immobilize”, as used herein, means to capture objects such as nucleicacids in a reversible or irreversible manner. Particularly, “immobilizedon the solid support material” means that the object or objects areassociated with the solid support material for the purpose of theirseparation from any surrounding media, and can be recovered, forexample, by separation from the solid support material at a later point.In this context, “immobilization” can comprise the adsorption of nucleicacids to glass or other suitable surfaces of solid materials asdescribed supra. Moreover, nucleic acids can be immobilized specificallyby binding to capture probes, wherein nucleic acids are bound toessentially complementary nucleic acids attached to a solid support bybase-pairing. In the latter case, such specific immobilization leads tothe predominant binding of target nucleic acids.

As used herein, “purification”, “isolation” or “extraction” of nucleicacids relate to the following: Before nucleic acids may be analyzed in adiagnostic assay by amplification, they typically have to be purified,isolated or extracted from biological samples containing complexmixtures of different components. For the first steps, processes may beused which allow the enrichment of the nucleic acids. After thepurification or isolation of the nucleic acids including the targetnucleic acids from their natural surroundings, analysis may be performedvia the simultaneous amplification and detection described herein.

“Simultaneously”, as used herein, means that two actions, such asamplifying a first and a second or more nucleic acids, are performed atthe same time and under the same physical conditions. In one embodiment,simultaneous amplification of the at least first and second targetnucleic acids is performed in one vessel. In another embodiment,simultaneous amplification is performed with at least one nucleic acidin one vessel and at least a second nucleic acid in a second vessel, atthe same time and under the same physical conditions, particularly withrespect to temperature and incubation time wherein the internal controlnucleic acid mentioned above is present each of said vessels.

“Target nucleic acid” is used herein to denote a nucleic acid in asample which should be analyzed, i.e. the presence, non-presence and/oramount thereof in a sample should be determined.

The “first target nucleic acid” and the “second target nucleic acid” aredifferent nucleic acids.

The term “fluid sample” refers to a material that may potentiallycontain an analyte of interest. The sample can be derived from anysource, in particular any biological source, such as a physiologicalfluid, including blood, saliva, ocular lens fluid, cerebrospinal fluid,sweat, urine, stool, semen, milk, ascites fluid, mucous, synovial fluid,peritoneal fluid, amniotic fluid, tissue, cultured cells, or the like.The fluid sample subjected to the process described herein can bepretreated prior to use, such as preparing plasma from blood, dilutingviscous fluids, lysis, or the like. Methods of treatment can involvefiltration, distillation, concentration, inactivation of interferingcomponents, the addition of reagents, and the like. A fluid sample maybe used directly as obtained from the source or used following apretreatment to modify the character of the sample. In some embodiments,an initially solid or semi-solid material is rendered liquid bydissolving or suspending it with a suitable liquid medium. The fluidsample is suspected to contain a certain target nucleic acid.

The term “reaction vessel” comprises, but is not limited to, tubes orthe wells of plates such as microwell, deepwell or other types ofmultiwell plates, in which a reaction for the analysis of the fluidsample such as e.g. reverse transcription or a polymerase chain reactiontakes place. The outer limits or walls of such vessels are chemicallyinert such that they do not interfere with the analytical reactiontaking place within. Preferably, the isolation of the nucleic acids asdescribed above is also carried out in a multiwell plate.

In this context, multiwell plates in analytical systems allow parallelseparation and analyzing or storage of multiple samples. Multiwellplates may be optimized for maximal liquid uptake, or for maximal heattransfer. A preferred multiwell plate for use in the context of thepresent invention is optimized for incubating or separating an analytein an automated analyzer. Preferably, the multiwell plate is constructedand arranged to contact a magnetic device and/or a heating device.

A “separation station” is a device or a component of an analyticalsystem allowing for the isolation of the solid support material from theother material present in the fluid sample. Such a separation stationcan e.g. comprise, but is not limited to, a centrifuge, a rack withfilter tubes, a magnet, or other suitable components. In a preferredembodiment of the invention, the separation station comprises one ormore magnets. Preferably, one or more magnets are used for theseparation of magnetic particles, preferably magnetic glass particles,as a solid support. If, for example, the fluid sample and the solidsupport material are combined together in the wells of a multiwellplate, then one or more magnets comprised by the separation station cane.g. be contacted with the fluid sample itself by introducing themagnets into the wells, or said one or more magnets can be brought closeto the outer walls of the wells in order to attract the magneticparticles and subsequently separate them from the surrounding liquid.

A “wash buffer” is a fluid that is designed to remove undesiredcomponents, especially in a purification procedure. Such buffers arewell known in the art. In the context of the purification of nucleicacids, the wash buffer is suited to wash the solid support material inorder to separate the immobilized nucleic acid from any unwantedcomponents. The wash buffer may, for example, contain ethanol and/orchaotropic agents in a buffered solution or solutions with an acidic pHwithout ethanol and/or chaotropic agents. Often the washing solution orother solutions are provided as stock solutions which have to be dilutedbefore use.

For downstream processing of the isolated nucleic acids, it can beadvantageous to separate them from the solid support material beforesubjecting them to amplification.

An “elution buffer” as used herein is a suitable liquid for separatingthe nucleic acids from the solid support. Such a liquid may be distilledwater or aqueous salt solutions, such as Tris buffers like Tris HCl, orHEPES, or other suitable buffers known to the skilled artisan. The pHvalue of such an elution buffer is in some embodiments alkaline orneutral. Said elution buffer may contain further components such aschelators like EDTA, which stabilizes the isolated nucleic acids byinactivation of degrading enzymes.

“Amplification reagents”, in the context of the invention, are chemicalor biochemical components that enable the amplification of nucleicacids. Such reagents comprise, but are not limited to, nucleic acidpolymerases, buffers, mononucleotides such as nucleoside triphosphates,oligonucleotides e.g. as oligonucleotide primers, salts and theirrespective solutions, detection probes, dyes, and more.

“Oligonucleotides” and “modified oligonucleotides” are components formedfrom a plurality of nucleotides as their monomeric units. The phosphategroups are commonly referred to as forming the internucleoside backboneof the oligonucleotide. The normal linkage or backbone of RNA and DNA isa 3′ to 5′ phosphodiester linkage. Methods for preparing oligomericcompounds of specific sequences are known in the art, and include, forexample, cloning and restriction of appropriate sequences and directchemical synthesis. In the process described above, the oligonucleotidesmay be chemically modified, i.e. the primer and/or the probe comprise amodified nucleotide or a non-nucleotide compound. The probe or theprimer is then a modified oligonucleotide.

The term “primer” is used herein as known to the expert skilled in theart and refers to oligomeric compounds, primarily to oligonucleotides,but also to modified oligonucleotides that are able to prime DNAsynthesis by a template-dependent DNA polymerase, i.e. the 3′-end of theprimer provides a free 3′-OH group to which further nucleotides may beattached by a template-dependent DNA polymerase establishing 3′- to5′-phosphodiester linkage whereby deoxynucleoside triphosphates are usedand whereby pyrophosphate is released.

A “probe” also denotes a natural or modified oligonucleotide. As knownin the art, a probe serves the purpose to detect an analyte oramplificate. In the case of the process described above, probes can beused to detect the amplificates of the target nucleic acids. For thispurpose, probes typically carry labels.

“Labels”, often referred to as “reporter groups”, are generally groupsthat make a nucleic acid, in particular oligonucleotides or modifiedoligonucleotides, as well as any nucleic acids bound theretodistinguishable from the remainder of the sample (nucleic acids havingattached a label can also be termed labeled nucleic acid bindingcompounds, labeled probes or just probes). Labels are in someembodiments fluorescent labels, which may be fluorescent dyes such as afluorescein dye, a rhodamine dye, a cyanine dye, and a coumarin dye.Useful fluorescent dyes include FAM, HEX, JA270, CAL635, Coumarin343,Quasar705, Cyan500, CY5.5, LC-Red 640, LC-Red 705.

Any primer and/or probe may be chemically modified, i.e. the primerand/or the probe comprise a modified nucleotide or a non-nucleotidecompound. The probe or the primer is then a modified oligonucleotide.

A method of nucleic acid amplification is the Polymerase Chain Reaction(PCR) which is well known to the skilled person, and disclosed, amongother references, in U.S. Pat. No. 4,683,202,

Other amplification reactions comprise, among others, the Ligase ChainReaction, Polymerase Ligase Chain Reaction, Gap-LCR, Repair ChainReaction, 3SR, NASBA, Strand Displacement Amplification (SDA),Transcription Mediated Amplification (TMA), and Q-amplification.

Automated systems for PCR-based analysis often make use of real-timedetection of product amplification during the PCR process in the samereaction vessel. Key to such methods is the use of modifiedoligonucleotides carrying reporter groups or labels, as described above.

The internal control nucleic acid described herein and comprising thesequence of SEQ ID NO 4 preferably exhibits the following propertiesrelating to its sequence:

-   -   a melting temperature from 55° C. to 90° C., more preferably        from 65° C. to 85° C., more preferably from 70° C. to 80° C.,        most preferably about 75° C.    -   a length of up to 500 bases or base pairs, more preferably from        50 to 300 bases or base pairs, more preferably from 100 to 200        bases or base pairs, most preferably about 180 bases or base        pairs    -   a GC content from 30% to 70%, more preferably from 40% to 60%,        most preferably about 50%.

In some embodiments, the internal control nucleic acid consists of SEQID NO 4 or its complement. As used herein, a “sequence” is the primarystructure of a nucleic acid, i.e. the specific arrangement of the singlenucleobases of which the respective nucleic acids consists. It has to beunderstood that the term “sequence” does not denote a specific type ofnucleic acid such as RNA or DNA, but applies to both as well as to othertypes of nucleic acids such as PNA or others. Where nucleobasescorrespond to each other, particularly in the case of uracil (present inRNA) and thymine (present in DNA), these bases can be consideredequivalent between RNA and DNA sequences, as well-known in the pertinentart.

Clinically relevant nucleic acids are often DNA which can be derivedfrom DNA viruses like Hepatitis B Virus (HBV), Cytomegalovirus (CMV) andothers, or bacteria like Chlamydia trachomatis (CT), Neisseriagonorrhoeae (NG) and others. In such cases, it can be advantageous touse an internal control nucleic acid consisting of DNA, in order toreflect the target nucleic acids properties.

Therefore, in some embodiments, said internal control nucleic acid isDNA.

On the other hand, numerous nucleic acids relevant for clinicaldiagnostics are ribonucleic acids, like the nucleic acids from RNAviruses such as Human Immunodeficiency Virus (HIV), Hepatitis C Virus(HCV), the West Nile Virus (WNV), Human Papilloma Virus (HPV), JapaneseEncephalitis Virus (JEV), St. Louis Encephalitis Virus (SLEV) andothers. The process described herein can be readily applied to suchnucleic acids. In this case, it can be advantageous to use an internalcontrol nucleic acid consisting of RNA, in order to reflect the targetnucleic acids properties. If both RNA and DNA are to be analyzed in theprocess described supra, it is the internal control nucleic acid mayadvantageously be RNA, as the internal control nucleic acid shouldideally mimic the most sensitive target of an assay involving multipletargets, and RNA targets usually have to be more closely controlled.

Thus, in some embodiments the internal control nucleic acid describedherein is RNA.

Since RNA is more prone to degradation than DNA due to influences suchas alkaline pH, ribonucleases etc., internal control nucleic acids madeof RNA are preferably provided as armored particles. Armored particlessuch as especially armored RNA are described in EP 910643. In brief, theRNA, which can be produced chemically or, preferably, heterologously bybacteria such as E. coli, is at least partially encapsulated in a viralcoat protein. The latter confers resistance of the RNA towards externalinfluences, in particular ribonucleases. It must be understood thatinternal control DNA can also be provided as a phage-packaged and thusprotected particle. Both encapsulated RNA and DNA are useful as internalcontrol nucleic acids in the context described herein. In someembodiments, RNA control nucleic acids are armored with the MS2 coatprotein in E. coli. In a further embodiment, DNA control nucleic acidsare armored using lambda phage GT11.

Typically, in amplification-based nucleic acid diagnostics, RNAtemplates are transcribed into DNA prior to amplification and detection.

Hence, in some embodiments of the process described herein, saidamplification reagents comprise a polymerase with reverse transcriptaseactivity, the process further comprising between step e. and step f. thestep of incubating in said reaction vessels said purified nucleic acidswith said one or more amplification reagents for a period of time andunder conditions suitable for transcription of RNA by said polymerasewith reverse transcriptase activity to occur.

A “polymerase with reverse transcriptase activity” is a nucleic acidpolymerase capable of synthesizing DNA based on an RNA template. It isalso capable of the formation of a double-stranded DNA once the RNA hasbeen reverse transcribed into a single strand eDNA. In some embodiments,the polymerase with reverse transcriptase activity is thermostable.

In the amplification of an RNA molecule by a DNA polymerase, the firstextension reaction is reverse transcription using an RNA template, and aDNA strand is produced. The second extension reaction, using the DNAtemplate, produces a double-stranded DNA molecule. Thus, synthesis of acomplementary DNA strand from an RNA template by a DNA polymeraseprovides the starting material for amplification.

Thermostable DNA polymerases can be used in a coupled, one-enzymereverse transcription/amplification reaction. The term “homogeneous”, inthis context, refers to a two-step single addition reaction for reversetranscription and amplification of an RNA target. By homogeneous it ismeant that following the reverse transcription (RT) step, there is noneed to open the reaction vessel or otherwise adjust reaction componentsprior to the amplification step. In a non-homogeneous RT/PCR reaction,following reverse transcription and prior to amplification one or moreof the reaction components such as the amplification reagents may beadjusted, added, or diluted, for which the reaction vessel has to beopened, or at least its contents have to be manipulated.

Reverse transcription is an important step in an RT/PCR. It is, forexample, known in the art that RNA templates show a tendency towards theformation of secondary structures that may hamper primer binding and/orelongation of the eDNA strand by the respective reverse transcriptase.Thus, relatively high temperatures for an RT reaction are advantageouswith respect to efficiency of the transcription. On the other hand,raising the incubation temperature also implies higher specificity, i.e.the RT primers will not anneal to sequences that exhibit mismatches tothe expected sequence or sequences. Particularly in the case of multipledifferent target RNAs, it can be desirable to also transcribe andsubsequently amplify and detect sequences with single mismatches, forexample, in the case of the possible presence of unknown or raresubstrains or subspecies of organisms in the fluid sample.

In order to benefit from both advantages described above, i.e. thereduction of secondary structures and the reverse transcription oftemplates with mismatches, in some embodiments the RT incubation iscarried out at more than one distinct temperature.

Therefore, in some embodiments, said incubation of the polymerase withreverse transcriptase activity is carried out at different temperaturesfrom 30° C. to 75° C., from 45° C. to 70° C., or from 55° C. to 65° C.

As a further important aspect of reverse transcription, long RT stepscan damage the DNA templates that may be present in the fluid sample. Ifthe fluid sample contains both RNA and DNA species, it can thus befavorable to keep the duration of the RT steps as short as possible, butat the same time ensuring the synthesis of sufficient amounts of eDNAfor the subsequent amplification and optional detection of amplificates.

Thus, in some embodiments the period of time for incubation of thepolymerase with reverse transcriptase activity is up to 30 minutes, 20minutes, 15 minutes, 12.5 minutes, 10 minutes, 5 minutes, or 1 minute.

In a further embodiment, the polymerase with reverse transcriptaseactivity and comprising a mutation is selected from the group consistingof

-   -   a) a CS5 DNA polymerase    -   b) a CS6 DNA polymerase    -   c) a Thermotoga maritima DNA polymerase    -   d) a Thermus aquaticus DNA polymerase    -   e) a Thermus thermophilus DNA polymerase    -   f) a Thermus flavus DNA polymerase    -   g) a Thermus filiformis DNA polymerase    -   h) a Thermus sp. sps17 DNA polymerase    -   i) a Thermus sp. Z05 DNA polymerase    -   j) a Thermotoga neapolitana DNA polymerase    -   k) a Termosipho africanus DNA polymerase    -   l) a Thermus caldophilus DNA polymerase

Particularly suitable for these requirements are enzymes carrying amutation in the polymerase domain that enhances their reversetranscription efficiency in terms of a faster extension rate.

Therefore, in some embodiments the polymerase with reverse transcriptaseactivity is a polymerase comprising a mutation conferring an improvednucleic acid extension rate and/or an improved reverse transcriptaseactivity relative to the respective wildtype polymerase.

In some embodiments, the polymerase with reverse transcriptase activityis a polymerase comprising a mutation conferring an improved reversetranscriptase activity relative to the respective wildtype polymerase.

Polymerases carrying point mutations that render them particularlyuseful in the context of the invention are disclosed in WO 2008/046612.In particular, in some embodiments polymerases to be used in the contextof the present invention are mutated DNA polymerases comprising at leastthe following motif in the polymerase domain:

T-G-R-L-S-S-XhrXbs-P-N-L-Q-N(SEQ ID NO 15); wherein Xb7 is an amino acidselected from SorT and wherein Xbs is an amino acid selected from G, T,R, K, or L, wherein the polymerase comprises 3′-5′ exonuclease activityand has an improved nucleic acid extension rate and/or an improvedreverse transcription efficiency relative to the wildtype DNApolymerase, wherein in said wildtype DNA polymerase Xbs is an amino acidselected from D, E or N.

Particularly useful are mutants of the thermostable DNA polymerase fromThermus species Z05 (described e.g. in U.S. Pat. No. 5,455,170), saidvariations comprising mutations in the polymerase domain as comparedwith the respective wildtype enzyme Z05. Especially useful is a mutantZ05 DNA polymerase wherein the amino acid at position 580 is selectedfrom the group consisting of G, T, R, K and L.

For reverse transcription using a thermostable polymerase, Mn2+ may beused as the divalent cation and is typically included as a salt, forexample, manganese chloride (MnCl2), manganese acetate (Mn(OAc)2), ormanganese sulfate (MnSO4). If MnCl2 is included in a reaction containing50 mM Tricine buffer, for example, the MnCl2 is generally present at aconcentration of 0.5-7.0 mM; 0.8-1.4 mM may be present when 200 mM ofeach dGTP, dATP, dUTP, and, dCTP are utilized; and 2.5-3.5 mM MnCl2 maybe present. Further, in some embodiments Mg2+ is used as a divalentcation for reverse transcription.

Since it is included in some embodiments to reverse-transcribe RNAtarget nucleic acids into eDNA while preserving the DNA target nucleicacids so both eDNA and DNA can be used for subsequent amplification, theinternally controlled process described herein is particularly usefulfor the simultaneous amplification and detection of target nucleic acidsderived from both organisms having an RNA or organisms having a DNAgenome. This advantage considerably increases the spectrum of differentorganisms, especially pathogens, that can be analyzed under identicalphysical conditions.

Therefore, in some embodiments the first and second target nucleic acidscomprise RNA and DNA.

The target of the amplification step can be an RNA/DNA hybrid molecule.The target can be a single-stranded or double-stranded nucleic acid.Although the most widely used PCR procedure uses a double-strandedtarget, this is not a necessity. After the first amplification cycle ofa single-stranded DNA target, the reaction mixture contains adouble-stranded DNA molecule consisting of the single-stranded targetand a newly synthesized complementary strand. Similarly, following thefirst amplification cycle of an RNA/eDNA target, the reaction mixturecontains a double-stranded eDNA molecule. At this point, successivecycles of amplification proceed as described above.

In some embodiments, the amplified target nucleic acids and theamplified internal control nucleic acid are detected during or after theamplification reaction in order to evaluate the result of the analysis.

It can be favorable to monitor the amplification reaction in real time,i.e. to detect the amplified target nucleic acids and the amplifiedinternal control nucleic acid during the amplification itself.

Therefore, in some embodiments, the probes, and in particular the probewith SEQ ID NO 1, are labeled with a donor fluorescent moiety and acorresponding acceptor fluorescent moiety.

The methods set out above are preferably based on Fluorescence ResonanceEnergy Transfer (FRET) between a donor fluorescent moiety and anacceptor fluorescent moiety. A representative donor fluorescent moietyis fluorescein, and representative corresponding acceptor fluorescentmoieties include LC-Red 640, LC-Red 705, Cy5, and Cy5.5. Typically,detection includes exciting the sample at a wavelength absorbed by thedonor fluorescent moiety and visualizing and/or measuring the wavelengthemitted by the corresponding acceptor fluorescent moiety. In the processaccording to the invention, detection is preferably followed byquantitating the FRET. Preferably, detection is performed after eachcycling step. Most preferably, detection is performed in real time. Byusing commercially available real-time PCR instrumentation (e.g.,LightCycler™ or TaqMan®), PCR amplification and detection of theamplification product can be combined in a single closed cuvette withdramatically reduced cycling time. Since detection occurs concurrentlywith amplification, the real-time PCR methods obviate the need formanipulation of the amplification product, and diminish the risk ofcross-contamination between amplification products. Real-time PCRgreatly reduces turn-around time and is an attractive alternative toconventional PCR techniques in the clinical laboratory.

The LightCycler™ instrument is a rapid thermal cycler combined with amicrovolume fluorometer utilizing high quality optics. This rapidthermocycling technique uses thin glass cuvettes as reaction vessels.Heating and cooling of the reaction chamber are controlled byalternating heated and ambient air. Due to the low mass of air and thehigh ratio of surface area to volume of the cuvettes, very rapidtemperature exchange rates can be achieved within the thermal chamber.

TaqMan® technology utilizes a single-stranded hybridization probelabeled with two fluorescent moieties. When a first fluorescent moietyis excited with light of a suitable wavelength, the absorbed energy istransferred to a second fluorescent moiety according to the principlesof FRET. The second fluorescent moiety is generally a quencher molecule.Typical fluorescent dyes used in this format are for example, amongothers, FAM, HEX, CY5, JA270, Cyan and CY5.5. During the annealing stepof the PCR reaction, the labeled hybridization probe binds to the targetnucleic acid (i.e., the amplification product) and is degraded by the 5′to 3′ exonuclease activity of the Taq or another suitable polymeraseduring the subsequent elongation phase. As a result, the excitedfluorescent moiety and the quencher moiety become spatially separatedfrom one another. As a consequence, upon excitation of the firstfluorescent moiety in the absence of the quencher, the fluorescenceemission from the first fluorescent moiety can be detected.

In both detection formats described above, the intensity of the emittedsignal can be correlated with the number of original target nucleic acidmolecules.

As an alternative to FRET, an amplification product can be detectedusing a double-stranded DNA binding dye such as a fluorescent DNAbinding dye (e.g., SYBRGREEN I® or SYBRGOLD® (Molecular Probes)). Uponinteraction with the double-stranded nucleic acid, such fluorescent DNAbinding dyes emit a fluorescence signal after excitation with light at asuitable wavelength. A double-stranded DNA binding dye such as a nucleicacid intercalating dye also can be used. When double-stranded DNAbinding dyes are used, a melting curve analysis is usually performed forconfirmation of the presence of the amplification product.

Molecular beacons in conjunction with FRET can also be used to detectthe presence of an amplification product using the real-time PCR methodsof the invention. Molecular beacon technology uses a hybridization probelabeled with a first fluorescent moiety and a second fluorescent moiety.The second fluorescent moiety is generally a quencher, and thefluorescent labels are typically located at each end of the probe.Molecular beacon technology uses a probe oligonucleotide havingsequences that permit secondary structure formation (e.g. a hairpin). Asa result of secondary structure formation within the probe, bothfluorescent moieties are in spatial proximity when the probe is insolution. After hybridization to the amplification products, thesecondary structure of the probe is disrupted and the fluorescentmoieties become separated from one another such that after excitationwith light of a suitable wavelength, the emission of the firstfluorescent moiety can be detected.

Thus, in some embodiment the process described herein uses FRET, whereinthe probes comprise a nucleic acid sequence that permits secondarystructure formation, wherein said secondary structure formation resultsin spatial proximity between said first and second fluorescent moiety.

Efficient FRET can only take place when the fluorescent moieties are indirect local proximity and when the emission spectrum of the donorfluorescent moiety overlaps with the absorption spectrum of the acceptorfluorescent moiety.

Thus, in some embodiments, said donor and acceptor fluorescent moietiesare within no more than 5 nucleotides of each other on said probe.

In a further embodiment, said acceptor fluorescent moiety is a quencher.

It is further advantageous to carefully select the length of theamplicon that is yielded as a result of the process described above.Generally, relatively short amplicons increase the efficiency of theamplification reaction. Thus, a preferred aspect of the invention is theprocess described above, wherein the amplified fragments comprise up to450 bases, preferably up to 300 bases, further preferably up to 200bases, and further preferably up to 150 bases.

The internal control nucleic acid described herein can serve as a“quantitative standard nucleic acid” which is apt to be and used as areference in order to quantify, i.e. to determine the quantity of thetarget nucleic acids. For this purpose, one or more quantitativestandard nucleic acids undergo all possible sample preparation stepsalong with the target nucleic acids. Moreover, a quantitative standardnucleic acid is processed throughout the method within the same reactionmixture. It must generate, directly or indirectly, a detectable signalboth in the presence or absence of the target nucleic acid. For thispurpose, the concentration of the quantitative standard nucleic acid hasto be carefully optimized in each test in order not to interfere withsensitivity but in order to generate a detectable signal also at veryhigh target concentrations. In terms of the limit of detection (LOD, seebelow) of the respective assay, the concentration range for the“quantitative standard nucleic acid” is in some embodiments 20-5000×LOD,in further embodiments 20-1000×LOD, and in yet further embodiments20-5000×LOD. The final concentration of the quantitative standardnucleic acid in the reaction mixture is dependent on the quantitativemeasuring range accomplished.

“Limit of detection” or “LOD” means the lowest detectable amount orconcentration of a nucleic acid in a sample. A low “LOD” corresponds tohigh sensitivity and vice versa. The “LOD” is usually expressed eitherby means of the unit “cp/ml”, particularly if the nucleic acid is aviral nucleic acid, or as IU/ml. “Cp/ml” means “copies per milliliter”wherein a “copy” is copy of the respective nucleic acid. IU/ml standsfor “International units/ml”, referring to the WHO standard.

A widely used method for calculating an LOD is “Probit Analysis”, whichis a method of analyzing the relationship between a stimulus (dose) andthe quanta!(all or nothing) response. In a typical quanta!responseexperiment, groups of animals are given different doses of a drug. Thepercent dying at each dose level is recorded. These data may then beanalyzed using Probit Analysis. The Probit Model assumes that thepercent response is related to the log dose as the cumulative normaldistribution. That is, the log doses may be used as variables to readthe percent dying from the cumulative normal. Using the normaldistribution, rather than other probability distributions, influencesthe predicted response rate at the high and low ends of possible doses,but has little influence near the middle.

The Probit Analysis can be applied at distinct “hitrates”. As known inthe art, a “hitrate” is commonly expressed in percent [%] and indicatesthe percentage of positive results at a specific concentration of ananalyte. Thus for example, an LOD can be determined at 95% hitrate,which means that the LOD is calculated for a setting in which 95% of thevalid results are positive.

An example of how to perform calculation of quantitative results in theTaqMan format based on an internal control nucleic acid serving as aquantitative standard nucleic acid is described in the following: Atiter is calculated from input data of instrument-corrected fluorescencevalues from an entire PCR run. A set of samples containing a targetnucleic acid and an internal control nucleic acid serving as aquantitative standard nucleic acid undergo PCR on a thermocycler using aspecified temperature profile. At selected temperatures and times duringthe PCR profile samples are illuminated by filtered light and thefiltered fluorescence data are collected for each sample for the targetnucleic acid and the internal control nucleic acid. After a PCR run iscomplete, the fluorescence readings are processed to yield one set ofdye concentration data for the internal control nucleic acid and one setof dye concentration data for the target nucleic acid. Each set of dyeconcentration data is processed in the same manner. After severalplausibility checks, the elbow values (CT) are calculated for theinternal control nucleic acid and the target nucleic acid. The elbowvalue is defined as the point where the fluorescence of the targetnucleic acid or the internal control nucleic acid crosses a predefinedthreshold (fluorescence concentration). Titer determination is based onthe assumptions that the target nucleic acid and the internal controlnucleic acid are amplified with the same efficiency and that at thecalculated elbow value equal amounts of amplicon copies of targetnucleic acid and internal control nucleic acid are amplified anddetected. Therefore, the (CTQS-CTtarget) is linear to log (targetcone/QS cone). In this context, QS denotes the internal control nucleicacid serving as a quantitative standard nucleic acid. The titer T canthen be calculated for instance by using a polynomial calibrationformula as in the following equation:T′=10(a(CTQS−CTtarget)2+b(CTQS−CTtarget)+c)

The polynomial constants and the concentration of the quantitativestandard nucleic acid are known, therefore the only variable in theequation is the difference (CTQS-CTtarget).

Further, the internal control nucleic acid can in some embodiments serveas a “qualitative internal control nucleic acid”. A “qualitativeinternal control nucleic acid” is particularly useful for confirming thevalidity of the test result of a qualitative detection assay: Even inthe case of a negative result, the qualitative internal control must bedetected, otherwise the test itself is considered to be inoperative.However, in a qualitative setup, it does not necessarily have to bedetected in case of a positive result. As a consequence, itsconcentration must be relatively low. It has to be carefully adapted tothe respective assay and its sensitivity. In some embodiments, theconcentration for the qualitative internal nucleic acid is in a range of1 copy per reaction to 1000 copies per reaction. In relation to therespective assay's limit of detection (LOD), its concentration is insome embodiments between the LOD of an assay and the 25 fold value ofthe LOD, in further embodiments between the LOD and 10×LOD. In yetfurther embodiments, it is between 2× and 10×LOD. In other embodiments,it is between 5× and 10×LOD, or it is 5× or 10×LOD.

In some embodiments, it can be advantageous to add different internalcontrol nucleic acids to a fluid samples, but to use only one of them—atleast the one comprising the sequence of SEQ ID NO 4—for amplificationand detection by adding only the primers with SEQ ID NO 2 and SEQ ID NO3 and the probe with SEQ ID NO 1.

In some embodiments of the process described herein, all steps areautomated. “Automated” means that the steps of a process are suitable tobe carried out with an apparatus or machine capable of operating withlittle or no external control or influence by an individual. Only thepreparation steps for the method may have to be done by hand, forinstance, storage containers have to be filled and put into place, thechoice of samples has to be performed by a human being and further stepsknown to the expert in the field, such as the operation of a controllingcomputer. The apparatus or machine may automatically add liquids, mixthe samples or carry out incubation steps at specific temperatures.Typically, such a machine or apparatus is a robot controlled by acomputer which carries out a program in which the single steps andcommands are specified.

DESCRIPTION OF THE FIGURES

FIG. 1:

Schematic depiction of the sample preparation workflow as used in anembodiment of the invention.

Arrows pointing down denote addition of a component or reagent to eachrespective well of the deepwell plate mentioned above, arrows pointingup their respective removal. These actions were performed manually insteps 2, 3, 4, 21 and 22, by the process head of the apparatus in steps10, 14, 16, 18, and 24, and by the reagent head of the apparatus insteps 5, 6, 7, 11, 15 and 19.

It has to be understood that the volumes used can be adjusted flexiblywithin the spirit of the invention, preferably at least about up to 30%of the disclosed values. In particular, in the case of step 2, thesample volume is preferably variable in order to take into account thedifferent types of fluid samples which may require more or less startingmaterial for obtaining proper results, as known by the artisan.Preferably, the range is from about 100 ul to about 850 ul. Morepreferably, it is about 100 ul, about 500 ul or about 850 ul.Preferably, the volume in the respective vessels is adjusted to anidentical total volume with the diluent in step 3. Preferably, as in thescheme shown in FIG. 1, the total volume adds up to about 850 ul.

EXAMPLE

This example describes the performance comparison of two differentoligonucleotide sets targeting SEQ ID NO 4 (set 2 containing SEQ ID NOs2, 3 and 5 (in MMx R2-SEQ ID NO 5), and set 3 containing SEQ ID NOs 2, 3and 1 (in MMx R2-SEQ ID NO 1), and a reference set 1 containing SEQ IDNOs 6, 7 and 8 (in MMx R2-Reference/SEQ ID NO 6) targeting SEQ ID NO 9.

In brief, in the depicted embodiment, realtime PCR is carried out on anRNA virus (HCV) using an internal control nucleic acid comprising thesequences of both SEQ ID NO 4 and SEQ ID NO 9. All samples wereprocessed and analyzed within the same experiment, i.e. on the samemultiwell plate.

The following samples were prepared and subsequently analyzed:

Reagent Manufacturer: HCV Secondary Standard, 18000 IU/ml Roche Plasmidcomprising SEQ ID NOs 4 & 9 Roche 3E11parts/mL

Suitable other types of standards or targets are known and available tothe skilled artisan.

The instruments listed in the following table were used according to theinstructions of the respective manufacturer:

Instrument Manufacturer cobas ® 6800/8800 process cell Roche DiagnosticsAG (Rotkreuz, CH) cobas ® 6800/8800 analytical cycler Roche DiagnosticsAG (Rotkreuz, CH)

For sample preparation the following reagents were used as diluents:

Reagent Manufacturer: BULK MP SPECIMEN DILUENT PMC Roche (MPSD) - IC/IQSStorage Buffer K3 EDTA Plasma, PCR neg. Roche

The following dilutions were prepared in advance and stored overnight(plasma dilutions at −60 to −90° C., BULK MP SPECIMEN DILUENT PMCdilutions at 2-8° C.):

Target Concentration Matrix HCV 10 IU/ml K3 EDTA plasma SEQ ID NO 46.0E+04 parts/ml BULK MP SPECIMEN DILUENT PMC (MPSD)

Each respective sample (500 ul) and each respective specimen diluent(350 ul) were pipetted manually into a deepwell plate. To each wellcontaining an HCV sample, 50 ul of the quantitative control nucleic acidmentioned above (3000 particles/sample) were manually added.

The respective control nucleic acid was stored in the following buffer:

IC/IQS - Storage Buffer, MPSD Conc. or pH Tris (mM) 10 EDTA (mM) 0.1Sodium Azide (w/v, %) 0.05 Poly rA RNA (mg/1) 20 pH 8

Sample preparation was performed on a Cobas® 6800/8800 process cell(Roche Diagnostics AG, Rotkreuz, C H), following the workflow accordingto the scheme depicted in FIG. 1 and using the following reagents:

Protease reagent Conc. or pH Tris (mM) 10 EDTA (mM) 1 Calcium Chloride(mM) 5 Calcium Acetate (mM) 5 Esperase (mg/ml) 80 Glycerin (w/v, %) 50pH 5.5

MGP Reagent Conc. or pH MPG Powder (mg/ml) 60 Tris (mM) 30 Methylparaben(w/v, %) 0.1 Sodium Azide (w/v, %) 0.095 pH 8.5

Lysis Reagent Conc. or pH Guanidine Thiocyanate (M) 4 Sodium Citrate(mM) 50 Polydocanol (w/v, %) 5 Dithiotreitol (w/v, %) 2 pH 5.8

Wash buffer Conc. or pH Sodium Citrate (mM) 7.5 Methylparaben (w/v, %)0.1 pH 4.1

Elution buffer Conc. or pH Tris (mM) 30 Methylparaben (w/v, %) 0.09 pH9.1

During the final sample preparation step (eluate cool down) the workingMaster Mixes (MMx), containing amplification reagents MMx R1 and MMx R2,were added manually to each well of a microwell plate. The eluates(containing the isolated nucleic acids) were then transferred by theinstrument from the p-plate to the micro-well plate and mixed with theMMx. The microwell plates were then sealed automatically and transferredmanually into the stand-alone analytical cycler for amplification anddetection.

The following master mixes (each consisting of the two reagents R1 andR2) were used:

For MMx R2-Reference/SEQ ID NO 6:

R1 Reagent Concentration/50 μl-PCR [μM] Water (PCR grade) Mn(Ac)₂ * 4H₂O(pH 6.1 adjusted with 3300 Acetic Acid) NaN3/Ri, buffered with 10 mMTris at 0.018 pH 7 [%]

R2 Reagent Concentration/50 μl-PCR [μM] DMSO [%]  5.400% NaN3/Ri,buffered with 10 mM Tris at  0.027% pH 7 [%] Potassium acetate pH 7.0120'000        Glycerol [%]  3.000% Tween 20 (%)  0.015% Tricine pH 8.060′000       NTQ21-46A - Aptamer  0.2222 UNG (U/uL) 0.2  dGTP 400    dATP 400     dCTP 4000     dUTP 800     10 mM Tris buffer for primer1.800 SEQ ID NO 10 (HCV primer) 0.100 SEQ ID NO 11 (HCV primer) 0.100SEQ ID NO 12 (HCV primer) 0.100 SEQ ID NO 7 (IC primer) 0.300 SEQ ID NO8 (IC primer) 0.300 probe storage buffer FAM/HEX 0.550 SEQ ID NO 13 (HCVprobe) 0.100 SEQ ID NO 14 (HCV probe) 0.100 SEQ ID NO 6 (IC probe) 0.100Z05-D Polymerase (U/uL) 40 (U/reaction) Water

For in MMx R2-SEQ ID NO 5:

R1 Reagent Concentration/50 μl-PCR [μM] Water (PCR grade) Mn(Ac)2 * 4H2O(pH 6.1 adjusted with 3300 Acetic Acid) NaN3/Ri, buffered with 10 mMTris at 0.018 pH 7 [%]

R2 Reagent Concentration/50 μl-PCR [μM] DMSO [%]  5.400% NaN3/Ri,buffered with 10 mM Tris at  0.027% pH 7 [%] Potassium acetate pH 7.0120'000        Glycerol [%]  3.000% Tween 20 (%)  0.015% Tricine pH 8.060′000       NTQ21-46A - Aptamer  0.2222 UNG (U/uL) 0.2  dGTP 400    dATP 400     dCTP 4000     dUTP 800     10 mM Tris buffer for primer1.800 SEQ ID NO 10 (HCV primer) 0.100 SEQ ID NO 11 (HCV primer) 0.100SEQ ID NO 12 (HCV primer) 0.100 SEQ ID NO 2 (IC primer) 0.300 SEQ ID NO3 (IC primer) 0.300 probe storage buffer FAM/HEX 0.550 SEQ ID NO 13 (HCVprobe) 0.100 SEQ ID NO 14 (HCV probe) 0.100 SEQ ID NO 5 (IC Probe) 0.100Z05-D Polymerase (U/uL) 40 (U/reaction) Water

For in MMx R2-SEQ ID NO 1:

R1 Reagent Concentration/50 μl-PCR [μM] Water (PCR grade) Mn(Ac)2 * 4H2O(pH 6.1 adjusted with 3300 Acetic Acid) NaN3/Ri, buffered with 10 mMTris at 0.018 pH 7 [%]

R2 Reagent Concentration/50 μl-PCR [μM] DMSO [%]  5.400% NaN3/Ri,buffered with 10 mM Tris at  0.027% pH 7 [%] Potassium acetate pH 7.0120'000        Glycerol [%]  3.000% Tween 20 (%)  0.015% Tricine pH 8.060′000       NTQ21-46A - Aptamer  0.2222 UNG (U/uL) 0.2  dGTP 400    dATP 400     dCTP 4000     dUTP 800     10 mM Tris buffer for primer1.800 SEQ ID NO 10 (HCV primer) 0.100 SEQ ID NO 11 (HCV primer) 0.100SEQ ID NO 12 (HCV primer) 0.100 SEQ ID NO 2 (IC primer) 0.300 SEQ ID NO3 (IC primer) 0.300 probe storage buffer FAM/HEX 0.550 SEQ ID NO 13 (HCVprobe) 0.100 SEQ ID NO 14 (HCV probe) 0.100 SEQ ID NO 1 (IC Probe) 0.100Z05-D Polymerase (U/uL) 40 (U/reaction) Water

For amplification and detection, the microwell plate was sealed with anautomated plate sealer in the Cobas® 6800/8800 process cell (see above),and the plate was manually transferred to the Cobas® 6800/8800analytical cycler (see above).

The amplification and detection (Real Time PCR) was carried outsimultaneously and under identical conditions for the three Master Mixesusing the generic PCR profile shown in Table 1. In total, sevenAmplification-Detection-Plates were run to obtain the requiredreplicates.

TABLE 1 Thermocycling profile Target Acquisition Plateau Measure RampRate [° C.] Mode [hh:mm:ss] [hh:mm:ss] [° C./s] Pre- UNG-Step 50 none00:02:00 00:00:00 2.2 PCR UNG/Template 94 none 00:00:05 00:00:00 4.4Denaturation RT-Step 55 none 00:02:00 00:00:00 2.2 60 none 00:06:0000:00:00 4.4 65 none 00:04:00 00:00:00 4.4 1st Measurement 95 none00:00:05 00:00:00 4.4 55 single 00:00:30 00:00:08 2.2 2nd Measurement 91none 00:00:05 00:00:00 4.4 58 single 00:00:25 00:00:08 2.2 Cooling 40none 00:02:00 00:00:00 2.2

TABLE 2 Cycles Overview Name Cycles Pre-PCR 1 1st Measurement 5 2ndMeasurement 45 Cooling 1

TABLE 3 Integration times Filter Combination Integration Time (sec)435-470 0.30 495-525 0.50 540-580 0.50 610-645 0.20 680-700 1.00

The Pre-PCR program comprised initial denaturing and incubation at 55,60 and 65° C. for reverse transcription of RNA templates. Incubating atthree temperatures combined the advantageous effects that at lowertemperatures slightly mismatched target sequences (such as geneticvariants of an organism) are also transcribed, while at highertemperatures the formation of RNA secondary structures is suppressed,thus leading to a more efficient transcription.

PCR cycling was divided into two measurements, wherein both measurementsapplied a one-step setup (combining annealing and extension). The first5 cycles at 55° C. allowed for an increased inclusivity bypre-amplifying slightly mismatched target sequences, whereas the 45cycles of the second measurement provided for an increased specificityby using an annealing/extension temperature of 58° C.

Using this profile on all samples comprised on the microwell platementioned above, amplification and detection was achieved in allsamples. This shows that the sample preparation prior to amplificationwas also successfully carried out.

The results show that the controls were also successfully amplified inall cases. The quantitation of the HCV target in the quantitative setupwas calculated by comparison with the respective internal controlnucleic acid serving as a quantitative standard.

Data Analysis

The raw data files of the Analytical Cycler (xml file) were analyzedusing the PARTS software. The current Cobas® 6800/8800 HCV analysistemplate was used for data calculation, as well as positive/negativecalling for the three master mixes in channel 4/JA270 (HCV signal) andchannel5/Cy5.5 (quantitative control signal). For HCV target andquantitative control the hit rates, CT, RFI and F-values were calculatedwith the current Cobas® 6800/8800 HCV analysis template

Results

The hit rates, CT-, RFI- and FValues for the three Master Mixes arelisted in Tables 4 and 5. The used analysis template was not optimizedfor each of the oligonucleotide sets.

TABLE 4 HCV Target CT values and RFI values with 500 uL sample processinput volume for EDTA Plasma Sample Comment Hit rate Hit rate (%) CTvalue RFI F Value 10 IU/mL HCV 85 of 100 85.00% average 40.03 13.731172262 MMx R2-Ref./SEQ STDEV 0.85 2.83 1122635 ID NO 6 CV 2.13% 20.61%95.77% 10 IU/mL HCV 90 of 97 92.78% average 40.56 9.10 477216 MMx R2-SEQID STDEV 1.16 2.19 626006 NO 5 CV 2.86% 24.09% 131.18% 10 IU/mL HCV 93of 100 93.00% average 40.21 10.01 641844 MMx R2-SEQ ID STDEV 0.88 2.16749487 NO 1 CV 2.19% 21.56% 116.77%

TABLE 5 Internal control CT values and RFI values with 500 uL sampleprocess input volume for EDTA Plasma Sample Comment Hit rate Hit rate(%) CT value RFI F Value MMx R2- 100 of 100 100.00% average 33.30 46.61179536 Reference/SEQ STDEV 0.67 8.57 69381 ID NO 6 CV 2.02% 18.40%38.64% 10 IU/mL HCV  97 of 100 97.00% average 33.84 3.27 121976 MMxR2-SEQ STDEV 0.46 0.18 70998 ID NO 5 CV 1.37% 5.54% 58.21% 10 IU/mL HCV100 of 100 100.00% average 33.64 11.42 188650 MMx R2-SEQ STDEV 0.31 0.86145133 ID NO 1 CV 0.92% 7.53% 76.93%

Summary Probe Comparison:

The three internal control oligonucleotide sets, compared in aside-by-side study, showed no significant difference in hit rate. Atrend towards a better RFI value combined with a lower base line withMMx R2-SEQ ID NO 1 (oligonucleotide set 3) was observed. This allows foran improved detection of the control nucleic acid comprising SEQ ID NO4. By using the probe with SEQ ID NO 1 instead of SEQ ID NO 5, assaysusing an internal control nucleic acid comprising SEQ ID NO 4 thusbecome more reliable and robust.

What is claimed is:
 1. An isolated oligonucleotide for improveddetection of a control nucleic acid comprising the sequence of SEQ ID NO4, consisting of SEQ ID NO: 1 and attached to a detectable label.
 2. Acomposition for amplification and improved detection of a controlnucleic acid comprising the sequence of SEQ ID NO 4, comprising theoligonucleotides consisting of SEQ ID NOs: 1, 2 and 3, wherein theoligonucleotide consisting of SEQ ID NO: 1 is attached to a detectablelabel.
 3. A kit for controlling the detection of multiple target nucleicacids, the kit comprising: a detection probe consisting of SEQ ID NO: 1and attached to a detectable label, a pair of amplification primersconsisting of SEQ ID NO: 2 and SEQ ID NO: 3, and a control nucleic acidcomprising SEQ ID NO:
 4. 4. The kit of claim 3 further comprisingamplification reagents comprising a distinct set of primers and a probefor each of the multiple target nucleic acids, and a nucleic acidpolymerase.
 5. The kit of claim 4 wherein the nucleic acid polymerasehas reverse transcription activity.